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- PDB-2l3f: Solution NMR Structure of a putative Uracil DNA glycosylase from ... -

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Basic information

Entry
Database: PDB / ID: 2l3f
TitleSolution NMR Structure of a putative Uracil DNA glycosylase from Methanosarcina acetivorans, Northeast Structural Genomics Consortium Target MvR76
ComponentsUncharacterized protein
KeywordsStructural genomics / Unknown function / NORTHEAST STRUCTURAL GENOMICS CONSORTIUM (NESG) / PSI-Biology / Protein Structure Initiative
Function / homology
Function and homology information


hypoxanthine DNA N-glycosylase activity / Hydrolases; Glycosylases; Hydrolysing N-glycosyl compounds / DNA repair
Similarity search - Function
Hypoxanthine-DNA glycosylase / Uracil DNA glycosylase superfamily / Uracil-DNA Glycosylase, subunit E / Uracil-DNA glycosylase-like domain / Uracil-DNA glycosylase-like / Uracil DNA glycosylase superfamily / Uracil-DNA glycosylase-like domain superfamily / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Hypoxanthine DNA glycosylase
Similarity search - Component
Biological speciesMethanosarcina acetivorans (archaea)
MethodSOLUTION NMR / simulated annealing
Model detailslowest energy, model 1
AuthorsAramini, J.M. / Hamilton, K. / Ciccosanti, C.T. / Wang, H. / Lee, H.W. / Rost, B. / Acton, T.B. / Xiao, R. / Everett, J.K. / Montelione, G.T. / Northeast Structural Genomics Consortium (NESG)
CitationJournal: To be Published
Title: Solution NMR Structure of a putative Uracil DNA glycosylase from Methanosarcina acetivorans, Northeast Structural Genomics Consortium Target MvR76
Authors: Aramini, J.M. / Hamilton, K. / Ciccosanti, C.T. / Wang, H. / Lee, H.W. / Rost, B. / Acton, T.B. / Xiao, R. / Everett, J.K. / Montelione, G.T.
History
DepositionSep 13, 2010Deposition site: BMRB / Processing site: RCSB
Revision 1.0Oct 20, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Feb 22, 2012Group: Structure summary
Revision 1.3May 1, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Uncharacterized protein


Theoretical massNumber of molelcules
Total (without water)19,1511
Polymers19,1511
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Uncharacterized protein


Mass: 19150.650 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methanosarcina acetivorans (archaea) / Gene: MA_0462 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pMgK / References: UniProt: Q8TTH3

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-13C HSQC
1313D 1H-15N NOESY
1413D 1H-13C NOESY aliphatic
1513D 1H-13C NOESY aromatic
1622D 1H-13C HSQC high resolution (L/V methyl stereoassignment)
1713D HNCO
1813D HN(CA)CO
1913D HNCA
11013D HN(CO)CA
11113D CBCA(CO)NH
11213D HN(CA)CB
11313D HBHA(CO)NH
11413D (H)CCH-TOCSY
11513D CCH-TOCSY
11613D (H)CCH-COSY
11713D HNHA
11813D HBHANH
11911D 15N T1 and T2
12012D 1H-15N hetNOE
1213NH J-modulation
1224NH J-modulation
NMR detailsText: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR ...Text: THE PROTEIN IS MONOMERIC BY GEL FILTRATION CHROMATOGRAPHY, STATIC LIGHT SCATTERING AND 15N T1/T2 RELAXATION. THE STRUCTURE WAS DETERMINED USING TRIPLE RESONANCE NMR SPECTROSCOPY. SPECTRA FOR BACKBONE AND SIDE CHAIN ASSIGNMENTS WERE OBTAINED ON A 1.7-MM MICROCRYOPROBE AT 600 MHZ. ALL NOESY DATA WERE ACQUIRED AT 800 MHZ USING A 5-MM CROYOPROBE. BACKBONE ASSIGNMENTS WERE MADE USING PINE, AND THE SIDE CHAIN ASSIGNMENTS WERE COMPLETED MANUALLY. AUTOMATIC NOESY ASSIGNMENTS WERE DETERMINED USING CYANA 3.0. BACKBONE (PHI/PSI) DIHEDRAL ANGLE CONSTRAINTS WERE OBTAINED FROM TALOSplus. COMPLETENESS OF NMR ASSIGNMENTS (EXCLUDING C-TERMINAL His5): BACKBONE, 98.5%, SIDE CHAIN, 91.0%, AROMATICS, 84.3%, STEREOSPECIFIC METHYL, 100%, STEREOSPECIFIC SIDE CHAIN NH2: 64.0%. FINAL STRUCTURE QUALITY FACTORS (FOR RESIDUES 1 TO 161, PSVS 1.4), WHERE ORDERED RESIDUES [S(PHI) + S(PSI) > 1.8] COMPRISE: 2-34,39-81,92-144,146-160: (A) RMSD (ORDERED RESIDUES): BB, 0.6, HEAVY ATOM, 1.1. (B) RAMACHANDRAN STATISTICS FOR ORDERED RESIDUES: MOST FAVORED, 89.1%, ADDITIONALLY ALLOWED, 10.8%, GENEROUSLY ALLOWED, 0.0%, DISALLOWED, 0.0%. (C) PROCHECK SCORES FOR ORDERED RESIDUES (RAW/Z-): PHI-PSI, -0.26/-0.71, ALL, -0.15/-0.89. (D) MOLPROBITY CLASH SCORE (RAW/Z-): 16.95/-1.38 (E) RPF SCORES FOR GOODNESS OF FIT TO NOESY DATA (RESIDUES 1 TO 161): RECALL, 0.951, PRECISION, 0.899, F-MEASURE, 0.924, DP-SCORE, 0.722. (F) NUMBER OF CLOSE CONTACTS PER 20 MODELS: 11. THE FINAL FIVE HISTIDINE RESIDUES IN THE C-TERMINAL AFFINITY TAG WERE NOT INCLUDED IN THE STRUCTURE CALCULATIONS AND HAVE BEEN OMITTED FROM THIS DEPOSITION.

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Sample preparation

Details
Solution-IDContentsSolvent system
10.94 mM [U-100% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 90% H2O/10% D2O90% H2O/10% D2O
21.15 mM [U-5% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 90% H2O/10% D2O90% H2O/10% D2O
30.80 mM [U-5% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 4 % C12E5 PEG, 90% H2O/10% D2O90% H2O/10% D2O
41.15 mM [U-5% 13C; U-100% 15N] MvR76, 20 mM MES, 100 mM sodium chloride, 5 mM calcium chloride, 10 mM DTT, 0.02 % sodium azide, 50 uM DSS, 7 % Polyacrylamide gel, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.94 mMMvR76-1[U-100% 13C; U-100% 15N]1
20 mMMES-21
100 mMsodium chloride-31
5 mMcalcium chloride-41
10 mMDTT-51
0.02 %sodium azide-61
50 uMDSS-71
1.15 mMMvR76-8[U-5% 13C; U-100% 15N]2
20 mMMES-92
100 mMsodium chloride-102
5 mMcalcium chloride-112
10 mMDTT-122
0.02 %sodium azide-132
50 uMDSS-142
0.80 mMMvR76-15[U-5% 13C; U-100% 15N]3
20 mMMES-163
100 mMsodium chloride-173
5 mMcalcium chloride-183
10 mMDTT-193
0.02 %sodium azide-203
50 uMDSS-213
4 %C12E5 PEG-223
1.15 mMMvR76-23[U-5% 13C; U-100% 15N]4
20 mMMES-244
100 mMsodium chloride-254
5 mMcalcium chloride-264
10 mMDTT-274
0.02 %sodium azide-284
50 uMDSS-294
7 %Polyacrylamide gel-304
Sample conditionspH: 6.5 / Pressure: ambient / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE8001
Bruker AvanceBrukerAVANCE6002
Varian INOVAVarianINOVA6003

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Processing

NMR software
NameVersionDeveloperClassification
CNS1.1Brunger, Adams, Clore, Gros, Nilges and Readrefinement
CYANA3Guntert, Mumenthaler and Wuthrichrefinement
CYANA3Guntert, Mumenthaler and Wuthrichstructure solution
AutoStructure2.2.1Huang, Tejero, Powers and Montelionerpf analysis
PSVS1.4Bhattacharya and Montelionestructure quality analysis
NMRPipe2.3Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
MolProbity3.18Richardsonstructure quality analysis
TopSpin2.1Bruker Biospincollection
TopSpin2.1Bruker Biospindata analysis
PdbStat5.1Tejero & Montelionepdb coordinate analysis
PINE1Bahrami, Markley, Assadi, and Eghbalniachemical shift assignment
Sparky3.112Goddarddata analysis
Sparky3.112Goddardpeak picking
TALOS+Shen, Cornilescu, Delaglio and Baxgeometry optimization
PALESPALES (Zweckstetter, Bax)rdc analysis
VnmrJ2.1Variancollection
VnmrJ2.1Variandata analysis
RefinementMethod: simulated annealing / Software ordinal: 1
Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2729 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 234 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (18.4 ...Details: THE FINAL STRUCTURES ARE BASED ON A TOTAL OF 2729 CONFORMATIONALLY-RESTRICTING NOE-DERIVED DISTANCE CONSTRAINTS AND 234 DIHEDRAL ANGLE CONSTRAINTS; 0 HYDROGEN BOND CONSTRAINTS (18.4 CONSTRAINTS PER RESIDUE, 5.4 LONG RANGE CONSTRAINTS PER RESIDUE, COMPUTED FOR RESIDUES 1 TO 161 BY PSVS 1.4). IN ADDITION, 210 RESOLVED N-H RESIDUAL DIPOLAR COUPLINGS FOR ORDERED RESIDUES OBTAINED FROM TWO ALIGNMENTS WERE INCLUDED IN ALL STRUCTURE CALCULATIONS. STRUCTURE DETERMINATION WAS PERFORMED ITERATIVELY USING CYANA 3.0. THE 20 STRUCTURES OUT OF 100 WITH THE LOWEST TARGET FUNCTION WERE FURTHER REFINED BY RESTRAINED MOLECULAR DYNAMICS/ENERGY MINIMIZATION IN EXPLICIT WATER (CNS) WITH PARAM19.
NMR constraintsNOE constraints total: 2729 / NOE intraresidue total count: 741 / NOE long range total count: 869 / NOE medium range total count: 442 / NOE sequential total count: 677
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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